Carboxin (IUPAC name: 5,6-dihydro-2-methyl-1,4-oxathiine-3-carboxanilide; CAS name: 5,6-dihydro-2-methyl-N-phenyl-1,4-oxathiin-3-carboxamide CAS; number: 5234-68-4) is a systemic agricultural fungicide and seed treatment agent. It is a respiratory toxin that prevents the oxidation of succinate by inhibiting the tricarboxylic acid cycle enzyme, succinate dehydrogenase (Sdh). This enzyme is composed of two subunits, a flavoprotein and an iron-sulphur protein (Ip), which together with two membrane-anchoring proteins make up succinate-quinone oxidoreductase. Carboxin is thought to act by preventing the transfer of electrons from succinate to ubiquinone through inhibiting the reoxidation of the high-potential S3 centre of the Ip subunit. Furthermore, Carboxin resistance in Ustilago maydis is known to be determined by a single amino acid residing in the Cys-rich cluster which ligates the S3 centre. A previous report indicated that insertion of the carboxin resistance (cbx R) gene into the Ustilago maydis genome impaired the pathogenic ability of the fungus towards Zea mays, the corn host, and that carboxin resistance did not significantly alter pathogenicity and was therefore a suitable marker for use in genetic analysis of U. maydis (Current Microbiology, Volume 44, Number, 2002, pp. 67-70).
Sensitivity (lack of resistance) to fungicides is a problem in commercial strains of mushrooms. Mutant strains, produced from known parent strains by UV irradiation followed by selection and having a genetically stable phenotype of insensitivity (resistance) to carboxin or benodanil, have now been prepared. The fungus Verticillium fungicola is pathogenic to mushrooms and is difficult to control. Certain fungicides, notably carboxin and benodanil, give some control over this pathogen, but these fungicides are phytotoxic to mushroom mycelium. If mushroom strains can be made less sensitive to one of these fungicides, fungicides can be used to control fungal diseases to which they are susceptible. U.S. Pat. No. 4,608,775 provided fungicide-resistant strains of the mushroom Agaricus bisporus. This prior reference selected an Agaricus bisporus strain against carboxin using UV irradiation but the gene resistant to carboxin was not identified. Toshikazu Irie et al. cloned genomic and cDNA fragments encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (EC 1.3.99.1) from the edible basidiomycetous fungus, Pleurotus ostreatus (Biochimica et Biophysica Acta 1396 (1998), 27-31). Furthermore, Toshikazu Irie et al. developed a selection marker gene for transformation of Pleurotus ostreatus by introducing a point mutation in a gene which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (Curr Genet, 2000, 37: 209-212).
Flammulina velutipes (synonym: Collybia velutipes; common names: velvet foot; winter mushroom) is an edible agaric that is available in early spring or late fall when few other mushrooms are. It often occurs in clusters and has a viscid smooth orange to brown cap and a velvety stalk that turns black in maturity and pallid gills. Flammulina velutipes is a popular traditional foodstuff with high nutritive value and enhances immune function and resists cancer cells. However, no studies have been done on the carboxin resistance gene specifically expressed in Flammulina velutipes against carboxin. There is still a need to develop a selection marker gene against carboxin that is specifically expressed in Flammulina velutipes. 